Abstract/Project Summary Heart disease continues to be the leading cause of morbidity and mortality in the United States while treatment options remain limited and largely ineffective. Therefore, a greater understanding of intracellular signaling in cardiomyocytes that underlies cardiac disease pathogenesis will aid in the development of novel therapies and drug targets for the treatment of heart disease. Palmitoylation is a reversible lipid modification that is catalyzed by the recently discovered aspartate-histidine-histidine- cysteine (zDHHC) family of palmitoyl transferases and has diverse effects on protein function, including playing critical roles in cellular signaling. Here, we will investigate the role of the palmitoyl transferase, Godz/Zdhhc3, in the heart. We discovered by yeast two-hybrid screening that Rho GDP dissociation inhibitor (RhoGDI) directly interacts with Godz and found that overexpression of Godz in cardiomyocytes enhances RhoGDI palmitoylation, indicating that RhoGDI is a novel Godz substrate. RhoGDI functions as the master homeostatic regulator of RhoGTPases, signaling molecules with pivotal roles in cardiac disease pathogenesis. We generated transgenic mice with cardiac-specific overexpression of Godz to study the role of Godz in vivo. Transgenic Godz mice develop congestive heart failure that is preceded by enhanced palmitoylation of RhoGDI in the heart and increased abundance and activity of all cardiac-expressed RhoGTPases, which are regulated by RhoGDI. We hypothesize that Godz plays instrumental roles in pathogenic signaling in cardiomyocytes by inducing RhoGTPase levels and activity through palmitoylation of RhoGDI. This proposal will test our hypothesis with the following aims: 1. To determine the role of Godz-mediated palmitoylation in the regulation of cardiac signaling and failure and 2. To determine the role of RhoGDI palmitoylation in RhoGTPase signaling and cardiac disease pathogenesis. The initial portion of this proposal will be carried out in the laboratory of the renowned molecular cardiology researcher, Dr. Jeffery Molkentin, where I will investigate how Godz regulates signaling in cardiomyocytes and determine the effects of cardiac-specific deletion of Godz on cardiac signal transduction, function, and propensity to disease (Aim 1). In my independent laboratory, I will extend these studies by utilizing mice with a knock-in mutation for the palmitoylation site of RhoGDI (RhoGDIC79S) to decipher the physiological role of RhoGDI palmitoylation in the regulation of cardiac RhoGTPase signaling (Aim 2). I will further investigate Godz-mediated palmitoylation and palmitoylation-dependent signaling in cardiac homeostasis and pathophysiology in my independent laboratory. This work will uncover mechanisms whereby dynamic palmitoylation regulates molecular signaling in the heart, which will place me at the forefront of this emerging field and drive my research program for years to come.